Hi fellow netters:
Stemming from the previous discussion of polymerases, I have a problem to
pose to the group. I have to modify a vector in which I remove a section
of DNA and would like to bring the ends together. Unfortunately, it is set
up using SacI and NheI both of which leave the following sticky ends:
5'
---------..
-------------.. 3' overhang left by SacI
3'
and 5'
----------..
------.. 5' overhang left by NheI
3'
I'd like to be able to join these two ends and retain at the very least the
SacI site. As I interpret the details of T4 Polymerase, T4 will only end
fill the NheI site in the presence of dNTPs. WITHOUT dNTPs T4 will remove
the 3' overhang of the SacI site.
Using T4 should, if I understand things correctly, give back the NheI site
at the expense of the SacI site. Correct? Unless I'm missing something T4
can *not* end-fill the SacI site? (It can't go 3'->5')
I'd like to be able to keep BOTH sites but now I'm beginning to wonder if
that can even be done without the aid of a SacI-NheI linker (most likely
home made!).
I'm made three attempts to fit these ends together and I'm at witts end!
I'm open to any (reasonable) suggestions! ;-)
Thanks in advance
David
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